Biosensor device and method

ABSTRACT

A biosensor apparatus for detecting a binding event between a ligand and receptor. The apparatus includes a biosensor surface and surface-bound two-subunit heterodimer complexes composed of first and second, preferably oppositely charged peptides that together form an α-helical coiled-coil heterodimer. The first peptide is attached to the biosensor surface, and the second peptide carries the ligand, accessible for binding by a ligand-binding agent. Binding of anti-ligand binding agent to the surface-bound ligand is detected by a suitable detector. A ligand-specific biosensor surface can be readily prepared from a universal template containing the first charged peptide, by addition of a selected ligand attached to the second peptide.

BIOSENSOR DEVICE AND METHOD

This application claims the priority of U.S. Provisional Application No.60/016,196 filed Apr. 25, 1996, and U.S. Provisional Application No.60/016,385, filed Apr. 25, 1996, which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to biosensors, and in particular, to abiosensor for measuring a binding event between a ligand and aligand-binding receptor, and to methods for producing such biosensors.

BACKGROUND OF THE INVENTION

Diagnostic tools used for detecting or quantitating biological analytestypically rely on ligand-specific binding between a ligand and areceptor. Ligand/receptor binding pairs used commonly in diagnosticsinclude antigen-antibody, hormone-receptor, drug-receptor, cell surfaceantigen-lectin, biotin-avidin, substrate/enzyme, and complementarynucleic acid strands. The analyte to be detected may be either member ofthe binding pair; alternatively, the analyte may be a ligand analog thatcompetes with the ligand for binding to the complement receptor.

A variety of devices for detecting ligand/receptor interactions areknown. The most basic of these are purely chemical/enzymatic assays inwhich the presence or amount of analyte is detected by measuring orquantitating a detectable reaction product, such as goldimmunoparticles. Ligand/receptor interactions can also be detected andquantitated by radiolabel assays.

Quantitative binding assays of this type involve two separatecomponents: a reaction substrate, e.g., a solid-phase test strip and aseparate reader or detector device, such as a scintillation counter orspectrophotometer. The substrate is generally unsuited to multipleassays, or to miniaturization, for handling multiple analyte assays froma small amount of body-fluid sample.

In biosensor diagnostic devices, by contrast, the assay substrate anddetector surface are integrated into a single device. One general typeof biosensor employs an electrode surface in combination with current orimpedance measuring elements for detecting a change in current orimpedance in response to the presence of a ligand-receptor bindingevent. This type of biosensor is disclosed, for example, in U.S. Pat.No. 5,567,301.

Gravimetric biosensors employ a piezoelectric crystal to generate asurface acoustic wave whose frequency, wavelength and/or resonance stateare sensitive to surface mass on the crystal surface. The shift inacoustic wave properties is therefore indicative of a change in surfacemass, e.g., due to a ligand-receptor binding event. U.S. Pat. Nos.5,478,756 and 4,789,804 describe gravimetric biosensors of this type.

Biosensors based on surface plasmon resonance (SPR) effects have alsobeen proposed, for example, in U.S. Pat. Nos. 5,485,277 and 5,492,840.These devices exploit the shift in SPR surface reflection angle thatoccurs with perturbations, e.g., binding events, at the SPR interface.Finally, a variety of biosensors that utilize changes in opticalproperties at a biosensor surface are known, e.g., U.S. Pat. No.5,268,305.

Biosensors have a number of potential advantages over binding assaysystems having separate reaction substrates and reader devices. Oneimportant advantage is the ability to manufacture small-scale, buthighly reproducible, biosensor units using microchip manufacturingmethods, as described, for example, in U.S. Pat. Nos. 5,200,051 and5,212,050.

Another advantage is the potentially large number of different analytedetection regions that can be integrated into a single biosensor unit,allowing sensitive detection of several analytes with a very smallamount of body-fluid sample. Both of these advantages can lead tosubstantial cost-per-test savings.

A key element in the manufacture of biosensors, particularly multi-assaybiosensors, is the placement of analyte-specific binding molecules orenzymes at desired locations on a biosensor surface. Ideally, it wouldbe desirable to construct a universal biosensor surface under rigorousmicrochip manufacturing conditions, but allow a variety of differentsurface-region formats to be achieved under less restrictivemanufacturing conditions, which at one extreme would allow an end userto tailor the universal chip to a unique multi-analyte format.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a biosensor apparatus fordetecting a binding event between a ligand and ligand-binding agent. Theapparatus has a biosensor surface, and two-subunit heterodimer complexescarried on the surface. The complexes are composed of first and second,preferably oppositely charged peptides that together form an α-helicalcoiled-coil heterodimer. The first peptide is attached to the biosensorsurface, and a ligand is covalently attached to the second peptide,accessible for binding by a ligand-binding agent. Binding of ananti-ligand agent to the ligand is detected by a suitable detector inthe apparatus.

The first peptide subunit may be attached to the biosensor surfacecovalently, e.g., through an oligopeptide spacer or a hydrocarbon-chainspacer, or may be bound to the biosensor surface through a stablenon-covalent linkage, e.g., a biotin/avidin binding pair. The biosensorsurface may include multiple regions, each having a different selectedligand attached to the second-subunit peptide.

In one general embodiment, the biosensor surface includes a monolayercomposed of hydrocarbon chains anchored at their proximal ends to thebiosensor surface, and having free distal ends defining an exposedmonolayer surface. The heterodimer complexes in this embodiment arepreferably embedded in the monolayer, and the ligands are disposed on ornear the monolayer surface. The monolayer may be formed on a metal,e.g., gold film, and may be composed of 8-22 carbon atom chains attachedat their proximal ends to the biosensor surface by a thiol linkage. Thechains have a preferred molecular density of about 3 to 5 chains/nm²,and the dielectric constant of the monolayer, in the presence of suchsolution but in the absence of such binding receptor, is preferably lessthan about 2.

In a biosensor apparatus designed for amperometric detection of bindingof a ligand-binding agent to the monolayer ligand, the biosensor surfaceis an electrode, and the monolayer, including the heterodimer complexesembedded in the monolayer, is sufficiently close-packed and ordered toform an effective barrier to current across the monolayer mediated by aredox ion species in an aqueous solution in contact with the monolayer.Binding of a ligand-binding agent to the ligand on the monolayer surfaceis effective to increase current across the monolayer, mediated by suchredox species. A chamber in the apparatus is adapted to contain anaqueous solution of redox species in contact with the monolayer, and thedetector includes a circuit for measuring ion-mediated current acrossthe monolayer, in response to binding events occurring between thereceptor and ligand.

In a biosensor apparatus designed for gravimetric detection of bindingof a ligand-binding agent to the surface-bound ligand, the biosensorsurface is a piezoelectric crystal. The detector functions to (i)generate a surface acoustic wave in the crystal and (ii) detect theshift in wave frequency, velocity, or resonance frequency of the surfaceacoustic wave produced by binding of ligand-binding agent to the ligand.

In a biosensor designed for optical surface plasmon resonance (SPR)detection of binding of a ligand-binding agent to the surface-boundligand, the biosensor surface is a transparent dielectric substratecoated with a thin metal layer on which the monolayer is formed, wherethe substrate and metal layer form a plasmon resonance interface. Thedetector functions to excite surface plasmons at a plasmon resonanceangle that is dependent on the optical properties of the metal film andattached monolayer, and to detect the shift in plasmon resonance angleproduced by binding of ligand-binding agent to the ligand.

In a biosensor designed for optical detection of binding of aligand-binding agent to the surface bound ligand, the detector functionsto irradiate the biosensor surface with a light beam, and detect achange in the optical properties of the surface layer, e.g., monolayerwith embedded heterodimer, produced by binding of ligand-binding agentto the ligand.

In another aspect, the invention includes a method for producing aligand-specific biosensor for use in a biosensor apparatus capable ofdetecting a binding event between a ligand and ligand-binding receptor.The method involves contacting together: (a) a biosensor electrodehaving a biosensor surface and a first heterodimer-subunit peptideattached to the biosensor surface, and (b) a second, preferablyoppositely charged peptide capable of binding to the first peptide toform a two-subunit α-helical coiled-coil heterodimer. The second peptidehas an attached ligand capable of binding specifically to aligand-specific agent. The contacting is effective attach ligands to thebiosensor surface. The biosensor surface may include first and seconddiscrete regions, where the second heterodimer subunit peptide in eachregion has a different attached ligand.

In one general embodiment of the method, the biosensor surface has amonolayer composed of hydrocarbon chains (i) anchored at their proximalends to the biosensor surface, and (ii) having free distal ends definingan exposed monolayer surface. The first peptide is embedded in themonolayer, and binding of the second peptide to surface-bound firstpeptide is effective to dispose the ligand preferably on or near themonolayer surface.

More generally, the invention provides a method of constructing an arrayof different, selected biological reagents attached to different,selected regions on an assay support surface. The method includesattaching molecules of a first heterodimer-subunit peptide to thesupport surface, effective to cover the different regions on the surfacewith the first peptide molecules. The subunit peptide has protectinggroups which when photo-released, allow the peptide to interact with asecond, preferably oppositely charged heterodimer-subunit peptide, toform a two-subunit α-helical coiled-coil heterodimer.

The surface is irradiated in a selected region of the surface underconditions effective to deprotect the first peptide in the irradiatedregion only, then contacted with a second subunit peptide carrying theassay reagent. This contacting is effective to attach the selectedreagent to the exposed region of the surface only. The above steps arerepeated for different selected regions and assay reagents, until thedesired array of different, selected biological reagents disposed atdifferent selected regions on an assay support surface is produced.

In one embodiment, the first subunit peptide contains amino acidresidues with one or more protected carboxyl groups, e.g., glutamategroups with nitrophenolate protecting groups.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show elements of a biosensor apparatus in accordancewith of the invention, illustrating the apparatus before (1A) and after(1B) binding of a ligand-binding agent to the biosensor surface in theapparatus;

FIGS. 2A-2C show helical wheel representations of (2A) terminal heptadsof two exemplary heterodimer-subunit peptides in a parallel α-helicalheterodimer configuration; (2B) terminal heptads of two exemplaryheterodimer-subunit peptides in an antiparallel α-helical heterodimerconfiguration; and (2C) helical wheel representations of specificpeptides in an α-helical heterodimer configuration;

FIGS. 3A-3E show schematic representations of adjacent heptads of twoheterodimer-subunit peptides in a parallel configuration comparing thestabilizing/destabilizing effects of charged residues at the e and gpositions in homodimers vs. heterodimers;

FIGS. 4A and 4B illustrate alternative methods for coupling an HSP1subunit peptide to a biosensor surface in a biosensor;

FIGS. 5A and 5B illustrate hydrocarbon-chain monolayers formed on abiosensor surface in a biosensor with an K-coil peptide alone embeddedin the monolayer (5A) and a K-coil/E-coil heterodimer embedded in themonolayer (5B);

FIG. 6 shows elements of an amperometric biosensor constructed inaccordance with one embodiment of the invention;

FIG. 7 shows the change in oxidation (solid circles) and reduction (opensquares) current as a function of time after addition of E-coil peptidesubunit to an electrode of the type illustrated in FIG. 5A containing anembedded K-coil peptide subunit;

FIG. 8 shows changes in oxidation of Fe(CN)₆ ³⁻ /⁴⁻ (open circles) andreduction (open squares) as a function of time after addition of PAKpeptide to an electrode containing di-saccharide ligands on aK-coil/E-coil lipid monolayer;

FIG. 9 shows changes in oxidation of Fe(CN) ₆ ³⁻ /⁴⁻ (open circles) andreduction (open squares) as a function of time after addition ofVerotoxin peptide to an electrode containing trisaccharide ligands on aK-coil/E coil lipid monolayer;

FIG. 10 shows elements of a gravimetric biosensor constructed inaccordance with an embodiment of the invention;

FIG. 11 shows elements of a surface plasmon resonance biosensorconstructed in accordance with an embodiment of the invention;

FIG. 12 shows elements of an optical biosensor constructed in accordancewith an embodiment of the invention;

FIGS. 13A-13C illustrate steps in the attachment of an assay reagent toan irradiated region of a biosensor surface, in accordance with a methodof the invention; and

FIG. 14 is a cross-sectional view of a portion of a multi-testamperometric biosensor constructed in accordance with an embodiment ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

I. Biosensor Apparatus

FIGS. 1A and 1B show a simplified schematic view of a biosensorapparatus 20 for detecting a binding event between a ligand and aligand-binding receptor, in accordance with the invention. The apparatusincludes a reaction chamber 22 defined in part by a substrate 24 whichhas a biosensor surface 26 within the chamber.

The biosensor surface has attached thereto, two-subunit heterodimercomplexes, such as complexes 28, each complex carrying a ligand, such asligands 30, which forms one of the two binding pairs of aligand/anti-ligand agent whose binding serves as the "trigger" of ameasurable biosensor event, as will be described below. FIG. 1B showsthe condition of the biosensor surface after binding of ligand-bindingagent, such as indicated at 34, to a portion of the ligands on thebiosensor surface.

According to an important feature of the invention, each heterodimercomplex, such as complex 28, includes a first peptide subunit, such assubunit 28a, which is attached to the biosensor surface, e.g., bycovalent attachment, and a second, preferably oppositely chargedsubunit, such as subunit 28b, to which the ligand is attached. The twopeptides are constructed, as will be detailed below, for self-assemblyinto stable, two-subunit alpha-helix coiled-coil heterodimer complexes,and when so assembled, serve to anchor the ligand on the biosensorsurface as shown.

The chamber includes at least one port or opening 32 for introducing asolution or suspension into the chamber. Where the biosensor has aclosed chamber, as here, the chamber may additionally include a vent oroutlet port. The analyte introduced into the chamber, i.e., the compoundor material to be assayed, will be either an anti-ligand binding agent,or a ligand or ligand analog which is capable of competing withsurface-bound ligand for binding to a ligand-binding agent. Theanalyte--i.e., the ligand, ligand analog or anti-ligand agent--may be infree molecule form or may be part of a complex, e.g., a cell ormacromolecular complex. Where the analyte is a ligand or ligand analog,the apparatus further includes a ligand-binding agent which may beintroduced with the analyte or may be present in the chamber, e.g.,immobilized on the chamber walls or present in dried, unbound formwithin the chamber.

The biosensor apparatus also includes a detector or detector means 36for detecting the presence and/or level or binding of ligand bindingagent to the surface ligands. A variety of detectors are describedbelow. For simplicity, the detector in FIG. 1 is illustratedschematically, and includes a beam source 38 for producing a beam 44, abeam detector 40, and a control unit 42 operatively connected to thebeam source and detector for measuring changes in the beam, e.g., beamintensity, in response to binding of ligand-binding agent tosurface-bound ligand, as illustrated in FIG. 1B.

A. Heterodimer Subunit Peptides

The heterodimer-subunit peptides employed in the biosensor invention aretwo non-identical, preferably oppositely charged polypeptide chains,typically each about 21 to about 70 residues in length, having an aminoacid sequence compatible with their formation into two-strandedα-helical heterodimeric coiled-coils. They are designated herein as HSP1(heterodimer-subunit peptide 1), and HSP2 (heterodimer-subunit peptide2). In the discussion below, HSP1 will refer to the peptide attached tothe biosensor surface in the biosensor, and HSP2, to the peptide havingan attached ligand. It will be understood that these designations referto the functional role played by the subunit peptide, not the actualpeptide sequence.

In aqueous medium, the isolated heterodimer-subunit peptides aretypically random coils. When HSP1 and HSP2 are mixed together underconditions favoring the formation of α-helical coiled-coil heterodimers,they interact to form a two-subunit α-helical coiled-coil heterodimericcomplex.

Peptides in an α-helical coiled-coil conformation interact with oneanother in a characteristic manner that is determined by the primarysequence of each peptide: The tertiary structure of an α-helix is suchthat 7 amino acid residues in the primary sequence correspond toapproximately 2 turns of the α-helix. Accordingly, a primary amino acidsequence giving rise to an α-helical conformation may be broken downinto units of 7 residues each, termed heptads. The heterodimer-subunitpeptides are composed of a series of heptads in tandem. When thesequence of a heptad is repeated in a particular heterodimer-subunitpeptide, the heptad may be referred to as a "heptad repeat", or simply"repeat".

Specific types of amino acid residues at defined positions in eachheptad act to stabilize the two-stranded α-helical coiled-coilheterodimeric structure or complex. The heterodimer peptides may alsocontain residues that can be reacted (either intra- or inter-helically)to stabilize the α-helical or coiled-coil nature of the polypeptides.One example of a stabilizing modification is the incorporation of lactambridges in the first and last (terminal) repeats of heterodimer-subunitpeptides, as detailed in PCT application WO CA95/00293 for "HeterodimerPolypeptide Immunogen Carrier Composition and Method", publication dateNov. 23, 1995, which is incorporated herein by reference.

The dimerization of HSP1 and HSP2 is due to the presence of a repeatedheptad motif of conserved amino acid residues in each peptide's primaryamino acid sequence. The individual positions in each heptad aredesignated by the letters a through g for HSP1, and a' through g' forHSP2, as shown in FIGS. 2A and 2B. Repeating heptad motifs havingappropriate amino acid sequences direct the HSP1 and HSP2 polypeptidesto assemble into a heterodimeric α-helical coiled-coil structure underpermissible conditions. The individual α-helical peptides contact oneanother along their respective hydrophobic faces, defined as the a and dpositions of each heptad.

HSP1 and HSP2 may assemble into a heterodimer coiled-coil helix(coiled-coil heterodimer) in either parallel or antiparallelconfigurations. In a parallel configuration, the two heterodimer-subunitpeptide helixes are aligned such that they have the same orientation(amino-terminal to carboxyl-terminal). In an antiparallel configuration,the helixes are arranged such that the amino-terminal end of one helixis aligned with the carboxyl-terminal end of the other helix, and viceversa.

Diagrams of the relative orientations of the a-g positions of twointeracting α-helices are shown in FIGS. 2A and 2B. FIG. 2A shows anend-on schematic of the first two turns (one heptad) of two exemplaryheterodimer-subunit peptides, EE and KK, arranged in a parallelconfiguration. FIG. 2B shows an end-on schematic of the sameheterodimer-subunit peptides arranged in an antiparallel configuration.

Heterodimer-subunit peptides designed in accord with the guidancepresented herein typically show a preference for assembling in aparallel orientation vs. an antiparallel orientation. For example, theexemplary peptides identified by SED ID NO:1 and SEQ ID NO:2 in theabove CA95/00293 PCT patent application, form parallel-configurationheterodimers as do other peptide sequences discussed in the PCTapplication. When attaching a ligand to HSP2, it is generally desirableto attach the ligand at or near the end of the peptide that will formthe distal end of the heterodimer. In particular, where the heterodimerforms a parallel configuration, the HSP1 peptide is preferably anchoredto the biosensor surface at its C terminus, and the ligand attached tothe HSP2 peptide at its N terminus.

In FIGS. 2A, 2B and 2C, amino acids are circled and indicated by theone-letter code, and consecutive amino acid positions are numbered andjoined by lines with arrow heads indicating the N-terminal to C-terminaldirection. Interactions between the two helixes are indicated by arrows.Wide arrows crossing between the helixes depict hydrophobic interactionsbetween the a and d positions of adjacent helixes.

Ionic interactions between the e and g positions of adjacent helixes areindicated as curving arrows above and below the nexus of the helixes. InFIGS. 2A and 2B, position e of peptide EE is a Gln in the first and lastheptad, and a Glu in the internal heptads. The (bottom) curving arrowdepicting ionic interactions with this position is drawn with a dashedline to indicate that ionic interactions are present between internalheptads of the helixes, but not between the first and last, or terminal,heptads. Lactam bridges in FIGS. 2A and 2B are indicated as aright-angle line between the f and b positions within each helix.

The hydrophobic interactions between the helixes are due to hydrophobicresidues at the a and d positions of the heterodimer-subunit peptides.Residues at these positions, effective to maintain the helixes incontact, include leucine, isoleucine, valine, phenylalanine, methionine,tryptophan, tyrosine, alanine and derivatives of any of the above. Otherresidues, including alanine, cysteine, serine, threonine, asparagine andglutamine may also occupy a or d positions in some heptads, so long asothers are occupied by hydrophobic residues.

Appropriate selection of the specific residues to occupy the a and dpositions is important. If the hydrophobic interactions are strong, asis the case, for example, between helixes containing Ile at one of thepositions and Leu at the other position, a significant fraction of thehelixes will form as homodimers at pH 7, even if like-charged residuesare present at the e and g positions to discourage homodimer formation.If, on the other hand, residues at the a and d positions are selectedsuch that the hydrophobic interactions are too weak (for example, Ala atboth positions), the helixes may not form coiled-coil dimers at all.Preferably, residue pairs are selected that promote the formation ≧95%heterodimers at pH 7. An exemplary pair of residues at the a and dpositions, that results in hydrophobic interactions conducive to ≧95%heterodimer formation at pH 7, comprises Leu at one of the positions andVal at the other position. These residues are present at the a and dpositions of exemplary heterodimer-subunit peptides.

Dimeric coiled-coil conformations of α-helixes are preferably alsostabilized by ionic interactions between residues at the e and gpositions of adjacent helixes, as is illustrated in FIGS. 3A and 3D. Ifeach helix of a dimer has a positively-charged residue at one position,for example, e, and a negatively-charged residue at the other position,for example, g, homodimer formation is favored (FIG. 3A; compare withheterodimer in FIG. 3B). However, if each helix has like-chargedresidues at both positions, then two oppositely-charged helixes willtend to associate into heterodimers (FIG. 3D), as opposed to forminghomodimers (FIG. 3C, 3E). The reader is referred to above-mentioned WO95/31480 for exemplary heterodimer sequences and methods of synthesis.

B. Ligand Attachment to the Biosensor Surface

As noted above, one of the two subunit peptides (HSP1) in theheterodimer is attached to the biosensor surface, and the second peptide(HSP2) contains a ligand intended to participate in an analyte-dependentligand/anti-ligand binding reaction. In both cases, the peptide issynthesized, or derivatized after synthesis, to provide the requisiteattachment function and ligand, respectively.

Considering the modification of HSP1, the peptide may be synthesized, ateither its N or C terminus, to carry additional terminal peptides thatcan function as a spacer between the biosensor surface and thehelical-forming part of the peptide. FIG. 4A shows an HSP1 peptideattached to a metal, e.g, gold, surface 46 through an polypeptide spacer48 terminating in a cysteine or methionine residue which provides forcovalent coupling to the surface through a thiolate linkage, understandard conditions (e.g., Dakkouri, A. S., et al., Langmuir (1996)12:2849-2852).

For HSP1 coupling to a glass or polymer surface, the C or N terminalresidue can be derivatized with a suitable activated functional groupthat allows direct coupling of the peptide end to a selected amine,acid, alcohol, or aldehyde group on the surface. These groups can beintroduced during solid phase synthesis according to standard methods,with other reactive side chains in the peptide being protected withsuitable protecting groups. Alternatively, the HSP1 peptide can beattached to the biosensor surface thorough a high-affinity bindingreaction, such as between a biotin moiety carried on the peptide and anavidin molecule covalently attached to the surface.

Where the heterodimer is embedded in a hydrocarbon-chain monolayer, asdescribed below, the spacer anchoring the HSP1 peptide to the biosensorsurface may be a hydrocarbon chain, such as spacer chain 52 anchoringHSP1 to biosensor surface 50 in FIG. 4B. The chain is preferably afractional length of the chains making up the bilayer, such that thedistal ends of the heterodimer peptides in the assembled monolayer areat or near the exposed surface of the monolayer. Thus, for example, ifthe monolayer is made up of 18-carbon chains, the spacer is preferably2-10 carbons in length, depending on the length of the assembledheterodimer.

The hydrocarbon-chain spacer, in the form of a omega-thio fatty acid,may be coupled to a terminal hydroxyl or amine coupling duringsolid-phase synthesis, as outlined above. The derivatized peptide, inturn, can be attached to a metal surface by standard thiolate coupling(Dakkouri, supra).

Considering the ligand-attachment to HSP2, the ligand selected will bedetermined by the analyte to be tested. Ligand-receptor binding pairs,i.e., ligand/ligand-binding agent pairs used commonly in diagnosticsinclude antigen-antibody, hormone-receptor, drug-receptor, cell surfaceantigen-lectin, biotin-avidin, substrate/enzyme, and complementarynucleic acid strands. The ligand is typically the smaller of the twobinding pair members, particularly where the ligand is attached to ahydrocarbon-chain monolayer, as described below. However, attachment ofeither binding pair is contemplated herein.

Where the ligand is a polypeptide, e.g., peptide antigen, the antigencan be synthesized by either solid-state or recombinant methods, toinclude the peptide antigen at the end of the HSP2 peptide that willorient distally in the assembled heterodimer. Where the ligand is anon-peptide moiety, e.g., a non-peptide hormone, drug, or nucleic acid,the HSP2 peptide can be synthesized to include one or more residues thatcan be specifically derivatized with the ligand. The ligand ispreferably covalently attached to an amino-acid coupling residues atpositions b, c and/or f of one or more heptads in HSP2 (FIG. 2A). Thesepositions lie along the outward face of a coiled-coil heterodimer. In anexemplary embodiment, a single coupling residue is placed at the fposition of a terminal heptad of HSP2, or at the terminal residue. Thisresidue may be derivatized during solid-state synthesis according toknown methods, allowing selective deprotection of the residue to bereacted.

Preferred coupling groups are the thiol groups of cysteine residues,which are easily modified by standard methods. Other useful couplinggroups include the thioester of methionine, the imidazolyl group ofhistidine, the guanidinyl group of arginine, the phenolic group oftyrosine and the indolyl group of tryptophan. These coupling groups canbe derivatized using reaction conditions known to those skilled in theart.

To attach the ligand-derivatized HSP2 peptide to the surface-immobilizedHSP1 peptide, the two peptides are contacted under conditions that favorheterodimer formation. A medium favoring coiled-coil heterodimerformation is a physiologically-compatible aqueous solution typicallyhaving a pH of between about 6 and about 8 and a salt concentration ofbetween about 50 mM and about 500 mM. Preferably, the salt concentrationis between about 100 mM and about 200 mM. An exemplary benign medium hasthe following composition: 50 mM potassium phosphate, 100 mM KCl, pH 7.Equally effective media may be made by substituting, for example, sodiumphosphate for potassium phosphate and/or NaCl for KCl. Heterodimers mayform under conditions outside the above pH and salt range, medium, butsome of the molecular interactions and relative stability ofheterodimers vs. homodimers may differ from characteristics detailedabove. For example, ionic interactions between the e and g positionsthat tend to stabilize heterodimers may break down at low or high pHvalues due to the protonation of, for example, Glu side chains at acidicpH, or the deprotonation of, for example, Lys side chains at basic pH.Such effects of low and high pH values on coiled-coil heterodimerformation may be overcome, however, by increasing salt concentration.

Increasing the salt concentration can neutralize the stabilizing ionicattractions or suppress the destabilizing ionic repulsions. Certainsalts have greater efficacy at neutralizing the ionic interactions. Forexample, in the case of the K-coil peptide in FIG. 2A, a 1M or greaterconcentration of ClO₄ ⁻ anions is required to induce maximal α-helicalstructure (as determined by CD measurements performed as detailed inExample 2), whereas a 3M or greater concentration of Cl⁻ ions isrequired for the same effect. The effects of high salt on coiled-coilformation at low and high pH also show that interhelical ionicattractions are not essential for helix formation, but rather, controlwhether a coiled-coil tends to form as a heterodimer vs. a homodimer.

C. Biosensor Surface with Hydrocarbon-Chain Monolayer

In one preferred embodiment, for use in a variety of the biosensorsdescribed below, the biosensor surface is modified to contain ahydrocarbon-chain monolayer, as illustrated in FIGS. 5A and 5B. Thefigures are enlarged views of a portion of a biosensor surface 48,including a thin electrode film 50 on a substrate 52, and a monolayer 54formed of hydrocarbon chains, such as chains 56, attached to the filmthrough thioether linkages. Embedded in the monolayer are molecules ofthe HSP1 peptide, such as molecules 58 (FIG. 5A, before addition of HSP2peptide), anchored to the surface as described above, and heterodimercomplexes, such as complexes 59 (FIG. 5B, after addition of HSP2peptides).

The chains forming the monolayer are typically 8-22 carbon, saturatedhydrocarbon chains, although longer chains, chains with someunsaturation, chains with non-carbon chain atoms, such as lipid ethers,and/or chains with minor branching, such as by non-chain methyl groups,may be employed. In an amperometric biosensor embodiment, to bedescribed below, the chains are sufficiently close packed and ordered toform an effective a barrier to electron transfer flow, under biosensoroperating conditions, as discussed below. This density is calculated tobe between 3-5 chains/nm².

With reference to FIG. 5A, the HSP1 peptide is included in the monolayerin a mole ratio peptide/hydrocarbon chains of preferably between 1:100to 1:5. As indicated in the figure, and discussed below, the FIG. 5Amonolayer is leaky to ion carriers, such as Fe(CN)₆ ³⁻, and as a result,gives a measurable detector current in the absence of analyte. Theleakiness of the membrane is presumably due to the disruption of themonolayer by charge-charge repulsion between the charged peptides in themonolayer, as shown, and a diminution of the electrostatic potentialbarrier in the monolayer.

With reference to FIG. 5B, addition of an oppositely charged HSP2peptide neutralizes the HSP1 peptide charges, with the result that themembrane assumes a low conductance property, as evidenced bysubstantially reduced current in the presence of charge carriers. Thisproperty of the biosensor surface will be described further below withrespect to FIGS. 7-9.

In a preferred method for forming the monolayer, a mixture ofthiol-containing chains and thiol-terminated HSP1 peptide, at a selectedmole ratio, is actively driven to the surface by applying a positivevoltage potential to the substrate surface, e.g., gold film.

In practice, the hydrocarbon chain mixture (about 1 mM hydrocarbonchains) in an ethanolic solution of 100 mM Li perchlorate, neutral pH,is placed over the electrode, and a selected potential is applied to theelectrode. The buildup of the monolayer can be monitored by increase inlayer thickness. Alternatively, monolayer formation is monitored bymeasuring current across the monolayer, as described below. In thiscase, formation of the monolayer will be characterized by a steady dropin electrode current, until minimum current is reached, at which pointmaximum chain packing has been achieved.

The time required to achieve saturation packing density will vary withapplied voltage, and can be as short as 10 seconds--about 4 orders ofmagnitude faster than monolayer formation by diffusion. Complete ornearly complete monolayer formation (30 Å thickness) occurs within 10minutes at about 1V potential and above. At lower positive voltages,additional reaction time is required. Preferably the voltage applied tothe electrode is at least between about +250 mV relative to a normalhydrogen electrode (+250 vs. NHE) and 1.2V (vs. NHE).

Not only are rapid monolayer formation times achieved, but thepercentages of peptide and hydrocarbon chains present in the reactionmixture are precisely represented in the monolayers, giving highlyreproducible electrode characteristics.

To complete formation of the monolayer with attached ligand, theligand-derivatized HSP2 peptide is contacted with the monolayer underconditions favoring heterodimer formation, as detailed above, where theHSP2 peptide is preferably added in excess. The formation ofheterodimers can be followed by measuring current across the monolayer.Because heterodimer formation tends to "tighten" the monolayer, asdiscussed above, heterodimer formation will lead to a steady drop inmeasured electrode current, until a stable low current is reached, atwhich point maximum heterodimer formation has occurred.

The subsections below illustrate several types of biosensors for whichthe biosensor surfaces described above are suitable.

D. Amperometric Biosensor

FIG. 6 illustrates, in simplified view, elements of an amperometricbiosensor 60 constructed in accordance with one embodiment of theinvention. The apparatus has a closed chamber 62 housing a biosensorsurface 64 formed of a gold film 66, which forms the surface electrodein the biosensor. The electrode surface is covered by a monolayer 66 ofhydrocarbon chains which define an exposed monolayer surface 68. Themonolayer includes ligand-bearing heterodimers, such as indicated at 70,embedded therein, with the ligands disposed at or near the monolayersurface, for accessibility to reaction with ligand-binding agents. Themonolayer is formed as above, e.g., with thioether attachment ofmonolayer components to the gold film.

The biosensor chamber serves to hold an aqueous electrolyte solutionrequired for biosensor operation, as will be described. Liquid may beintroduced into or withdrawn from the chamber through a valved port 72,and chamber may include a second port or vent (not shown) to facilitateliquid flow through the port.

A reference electrode 74 and a counter electrode 76 in the apparatus arecarried on the upper chamber wall, as shown, and are both in conductivecontact with electrode 64 when the chamber is filled with electrolytesolution. The reference electrode, which is held at ground, serves asthe voltage potential reference of the working electrode, when aselected potential is placed on the working electrode by a voltagesource 78. This potential is measured by a voltage measuring device 80which may additionally include conventional circuitry for maintainingthe potential at a selected voltage, typically between about -500 to+800 mV.

Voltage source 78 is connected to counter electrode 76 through a currentmeasuring device 82 as shown, for measuring current between the twoelectrodes during biosensor operation. The reference and counterelectrodes are Pt, Ag, Ag/AgCl, or other suitable electrodes. Thereference and working electrodes, and the circuitry connecting them tothe working electrode, are also referred to herein, collectively, asdetector means for measuring ion-mediated current across theworking-electrode monolayer, in response to ligand-receptor bindingevents occurring at the monolayer surface.

In operation, the chamber is filled with a solution containing analyteand ionic species capable of undergoing a redox reaction, i.e., losingor gaining an electron, at a suitably charged electrode. Exemplary redoxspecies are Fe (CN)₆ ^(3-/4-), as a negatively charged species, andRu(NH₃)₆ ^(2+/3+) as a positively charged species. Other probes whichcan be used include Mo(CN)₆ ³⁻ (E₀ =+800 mV), W(CN)₆ ³⁻ (E₀ =+580 mV),Fe(CN)₄ ⁻ (E₀ =+580 mV), Ce^(4+/3+) (E₀ =+1.4V) , and Fe^(+3/2+) (E₀=+666 mV) Typical redox ion concentrations are between 0.01 and 10 mM.The redox solution is contained in chamber and is in contact withreference and counter electrodes.

The voltage potential placed on the electrode, i.e., between theelectrode and reference electrode, is typically at least 90 mV above theelectrochemical potential (E₀) value of the redox species, foroxidation, and at least 90 mV below the electrochemical potential, forreduction of the species. Consider, for example, Fe (CN)₆ ^(3-/4-), withan E₀ of 450 mV (vs. NHE) Above about 550 mV electrode potential, anyFe2+ species is oxidized to Fe3+, and at an electrode potential belowabout 350 mV, and Fe+3 is reduced to Fe+2. Similarly, Ru(NH₃)₆ ^(2+/3+)has an E₀ of +50 mV (vs. NHE), so oxidation is achieved at an electrodepotential above about +150 mV, and reduction, below about -50 mV.

The ability of heterodimer formation in the monolayer to enhance theclose packed structure of the monolayer, as evidenced by monolayerconductance, is illustrated in FIG. 7. The figure shows the drop inconductance, as measured by ion-mediated current flow, after addition toa monolayer containing a K-coil peptide alone, of an oppositely chargedE-coil peptide. The pairing of the two peptides to form charge-neutralheterodimers in the monolayer is effective to reduce monolayerconductance substantially, as evidenced by the time-dependent fall inmeasured oxidation or reduction current in the presence of Fe(CN)₆ ³⁻ions.

In one exemplary biosensor, the biosensor surface includes (i) amonolayer with embedded K-coil peptides (HSP1) covalently attached tothe electrode surface, (ii) oppositely charged E-coil peptides (HSP2)forming heterodimers with the K-coil peptides in the monolayer, and(iii) surface disaccharide ligands derivatized to the E-coil peptidesand disposed therefore at the monolayer surface. As seen in FIG. 8,addition of the anti-ligand receptor (a PAK protein receptor) producesan increase in both oxidation and reduction currents, with the currentincrease over time reflecting the kinetics of receptor binding to thesurface ligands.

A similar biosensor having a trisaccharide, rather than disaccharide,ligand attached to the E-coil peptide subunit in the electrode monolayerwas tested with a Verotoxin receptor, with the results seen in FIG. 9.The solid lines in the figure show the increase in oxidation andreduction current observed, as a function of time, after addition ofVerotoxin.

In the absence of receptor binding to the ligand, the monolayer retainsits dense ordered packing, forming an effective barrier to currentacross the monolayer mediated by the redox ion species, when a suitableoxidizing or reducing potential is placed across the monolayer. This isreflected by a low or zero measured current across the membrane. Thedielectric constant of the monolayer in this condition is typicallyabout 1-2.

The triggering event in the biosensor is the binding of a ligand-bindingagent to the surface-bound ligand. This binding perturbs the orderedstructure of the monolayer sufficiently to allow the movement of redoxspecies through the monolayer, producing current through the electrode.Measurements performed in support of the invention indicate that onetriggering event leads to 10² to 10⁶ ionic and electron transfer eventsper second, and thus is highly multiplicative. The biosensor recordsthis binding event as an increase in current across the electrode, i.e.,between the working and counter electrodes.

By analogy to a transistor, the redox solution serves as the "source",the monolayer as the "gate", and the underlying electrode as the"drain". Current in a transistor is initiated by applying a thresholdvoltage to the gate. In the biosensor of the invention, current isinitiated by a stimulus--in this case, a ligand-receptor bindingevent--to the monolayer "gate".

E. Gravimetric Biosensor

FIG. 10 shows basic elements of a gravimetric biosensor 86 incorporatingthe novel biosensor surface of the invention. The biosensor has apiezoelectric crystal 90 whose biosensor surface 92 includes a monolayer94 with ligand-bearing heterodimer complexes, such as complex 96,embedded therein.

Surface acoustic waves (SAW) are generated in the crystal by anoscillator 96. According to known piezoelectric biosensor principles,the change in mass in the biosensor surface resulting from the bindingof ligand-binding agent to the surface-bound ligand alters thefrequency, resonance frequency, and wavelength of the SAW, and at leastone of these wave characteristics is measured by a detector 98. Theoscillator and detector collectively form detector means for detectingbinding of ligand-binding agent to the biosensor surface. Details ofcrystal construction and associated detector means in gravimetricbiosensors are given, for example, in U.S. Pat. Nos. 5,478,756 and4,789,804, and in PCT application WO 96/02830.

F. Surface Plasmon Resonance Biosensor

FIG. 11 shows basic elements of a surface plasmon resonance (SPR)biosensor 100 incorporating the novel biosensor surface of theinvention. An open-top chamber 102 in the biosensor contains a waveguide104 composed of a dielectric film 106 and a thin evaporated metal film108 constructed to support surface plasmon waves at the dielectric/metalfilm interface. The waveguide surface forms a biosensor surface having amonolayer 110 with ligand-bearing heterodimer complexes, such as complex112, embedded therein.

A light source 114 direct a divergent light beam onto the biosensorsurface through a lens 116. At some region along the length of thebiosensor surface, the beam angle strikes the surface at an absorptionangle at which absorption from the evanescent wave by surface plasmonsoccurs. The absorption angle will shift with changes in the compositionof the material near the interface, that is, in response to bindingevents occuring on the monolayer surface.

The intensity of reflected light from each region along the biosensorsurface is monitored by a photosensor 118 whose photosensing grid ismatched to specific detector surface regions, and which is operativelyconnected to an analyzer 120. The light source and photosensor are alsoreferred to herein as biosensor means.

In operation, the SPR absorption angle on the biosensor surface ismeasured before and after analyte addition, with the measured shift inangle being proportional to the extent of surface ligand binding toligand-binding agent.

G. Optical Biosensor

A variety of biosensor devices which rely on changes in the opticalproperties of a biosensor surface, in response to ligand/anti-ligandbinding events, have been proposed. FIG. 12 shows basic elements of anoptical biosensor apparatus 122 having an open chamber 124 and abiosensor surface 126 which includes a hydrocarbon-chain monolayer 128with embedded heterodimer complexes, such as shown at 130.

The detector means in the apparatus for detecting binding events on thebiosensor surface includes a source 132 of polarized light and a lenssystem 134 for directing the light in a beam through the region of themonolayer. A photodetector 136 at the opposite side of the biosensorsurface functions to measure intensity of light at a given polarizationangle, through a polarization filter 138. Detection of ligand bindingevents is based on the change of polarization angle and intensity oflight transmitted by the monolayer in response to perturbation of theregular order of the monolayer by surface binding events. These changesare recorded by an analyser 140 operatively connected to thephotosensor.

The biosensors described above have single-region biosensor surfaces,i.e., biosensor surfaces containing a single ligand, for use indetecting a single analyte. These surface are readily constructed, asdiscussed above, by contacting a selected HSP2-ligand conjugate with auniversal HSP1 biosensor surface, then adding the desired HSP2-ligandconjugate under conditions of heterodimer formation.

It will be appreciated that the method of the invention can be used toconstruct a biosensor with multiple sensor surfaces, or to partition asingle surface into several different-ligand regions, for carrying outmulti-analyte tests. In the latter embodiment, different HSP2-ligandconjugates are contacted with the different selected regions on auniversal sensor surface. The present invention allows for flexibilityin terms of number and types of ligands attached, after manufacture ofthe biosensor surface(s), particularly where the distinct biosensorregions can be selectively contacted with different HSP2-ligandconjugates.

The next section describes a more general method in accordance with theinvention for forming a biosensor surface with multi-reagent regions,for use particularly in constructing a biosensor surface with a highdensity of different ligand-containing test regions.

II. Producing a Multi-Ligand Surface

FIGS. 13A-13C illustrate the first iteration in a method of constructingan array of different biological reagents in different, selected regionson an assay support surface, in accordance with the invention.

FIG. 13A shows a portion of an assay support surface 142--in this case,a biosensor surface for use in an amperometric biosensor device. Thesurface has been constructed, e.g., by conventional photolithographicmethods, to include an array of electrode regions, such as the tworegions 144, 146. In the embodiment illustrated, the regions are metal,e.g., gold, film region formed on a substrate 148. Although not shownhere, the surfaces are prepared as above as hydrocarbon-chain monolayerswith HSP1 peptides, such as shown at 150, embedded in the monolayer.

According to an important feature of the invention, the HSP1 peptideshave one or more photo-releasable blocking groups, such as blockinggroups 150a on peptide 150, that prevent heterodimer formation in thepresence of HSP2 peptide under selected conditions. In the present case,the HSP1 peptide is an E-coil peptide, and the blocking groups arenitrophenolate protecting groups on two or more of the glutamate sidechain carboxyl groups. Those skilled in the art will recognize that avariety of photo-releasable blocking groups, e.g., variousphoto-deprotectable groups on one or more of amino acid side chains, canbe used to block heterodimer formation, either by steric interference orby reducing charge interactions.

Following attachment of the HSP1 peptide with blocking groups to theassay surface, or as part of a monolayer on the surface, the surface isselectively irradiated to release blocking groups in irradiated regionsof the surface only. This can be accomplished, as illustrated in FIG.13A, by irradiating the surface through a photomask 152 placed over thesurface, to selectively irradiate regions of the surface correspondingto photomask openings. FIG. 13B shows selective release of blockinggroups from the irradiated region 144.

The surface is then reacted with an HSP2 peptide 154 (FIG. 13C)conjugated to a selected ligand, as shown, to form heterodimersselectively in the unblocked regions of the surface. If necessary, theheterodimer formation conditions are selected, e.g., in ionic strength,to heterodimer formation with unblocked HSP1 peptides only. Thus, if thereleased blocking groups expose ionic groups, e.g., carboxyl groups, itmay be useful to lower the ionic strength of the reaction medium, toenhance ionic interaction effects leading to heterodimer formation. Theabove steps are repeated for each ligand to be added to the assaysurface, until the desired array of different ligands at differentaddressable regions of the surface is constructed.

FIG. 14 shows a portion of an multi-analyte assay surface constructedaccording to the above method, and employed in an amperometric biosensorapparatus 156 of the type described above. A biosensor surface 158 in achamber 157 has a plurality of independent sensor regions, such asregions 160, 162, each having a separate ligand, such as indicated atL₁, L₂, attached to the respective sensor region through heterodimers,such as heterodimers 166 (L₁) and 168 (L₂). The heterodimers areembedded in a monolayer on each region, such as monolayer 154, fordetection of different analytes in a analyte-containing sampleintroduced into the biosensor chamber.

Current flow in each detector region, such as regions 160, 162 isinterrogated by a multiplexer 172 which connects each region to thechamber reservoir through a voltage source 174 and current device 176.As above, binding of ligand-binding agent to any region will perturb themonolayer structure of that region, causing a measured current increasein the region(s) where such binding has occurred.

From the foregoing, it can be seen how various objects and advantages ofthe invention are met. The biosensor surface of the invention can beformed under controlled manufacturing conditions consistent withmicrochip scale and photomask processes, to produce highly uniformand/or miniaturized and/or high-density array sensor devices withattached HSP1 peptides.

After manufacture of a device with a universal surface, the sensorsurface can be readily adapted to a wide variety of ligand(s), byreacting the sensor surface with the an HSP2 peptide derivatized withthe selected ligand. The ligand-attachment reaction can be carried outunder relatively simple production conditions, and may even beaccomplished by the end user, thus combining both manufacturingprecision at the initial production stage, and assay flexibility at theligand-addition stage.

The invention is particularly useful in producing biosensor deviceswhich use or require close-packed monolayer biosensor surfaces, as inthe case of an amperometric biosensor. The studies reported in FIGS. 7-9show that formation of heterodimers in a hydrocarbon-chain monolayer arecompatible with a close-packed monolayer structure that forms aneffective barrier to ion-carrier movement, and at the same time, isresponsive to binding by a ligand-binding agent, to increase ion-carriermovement through the monolayer.

The invention is easily adapted to any of a variety of biosensordevices, such as those illustrated above. Further, the invention can bereadily adapted to producing multi-ligand biosensors, by selectivelycontacting different regions of a universal biosensor surface withdifferent selected HSP2-ligand conjugates.

In another aspect, the invention can be used to create multi-ligandassay surfaces by photomasking techniques that are capable of producinghighly reproducible microarray biosensor devices having a plurality ofdifferent-ligand regions.

Although the invention has been described with respect to particulardevices and methods, it will be understood that various changes andmodifications can be made without departing from the invention, asencompassed by the accompanying claims.

It is claimed:
 1. A biosensor apparatus for detecting a binding eventbetween a ligand and ligand-binding agent, comprisingmeans defining abiosensor surface, carried on said biosensor surface, two-subunitheterodimer complexes composed of first and second peptides thattogether form an α-helical coiled-coil heterodimer, where said firstpeptide is attached to the biosensor surface, a ligand covalentlyattached to the second peptide in the two-subunit heterodimer complexes,accessible for binding by a ligand-binding agent, means for introducingonto the biosensor surface, an analyte selected from the groupconsisting of a ligand-binding agent, and a ligand or ligand analogcapable of competing with a ligand-binding agent for binding to saidligand, said apparatus also including a ligand-binding agent when theanalyte is a ligand or ligand analog, and means for detecting thebinding of a ligand-binding agent to the ligand on the biosensorsurface.
 2. The apparatus of claim 1, wherein said biosensor surfaceincludes a monolayer composed of hydrocarbon chains anchored at theirproximal ends to the biosensor surface, and having free distal endsdefining an exposed monolayer surface, said heterodimer complexes areembedded in said monolayer, and the ligands are disposed on or near theexposed monolayer surface.
 3. The apparatus of claims 2, wherein theelectrode has a gold biosensor surface, said monolayer is composed of8-22 carbon atom chains attached at their proximal ends to the biosensorsurface by a thiol linkage, and said chains have a molecular density ofabout 3 to 5 chains/nm².
 4. The apparatus of claim 2, wherein the firstpeptide subunit is covalently attached to the biosensor surface-throughan oligopeptide spacer or a hydrocarbon-chain spacer.
 5. The apparatusof claim 2, designed for amperometric detection of binding of aligand-binding agent to the monolayer ligand, whereinthe biosensorsurface is an electrode, the monolayer, including the heterodimercomplexes, is sufficiently close-packed and ordered to form an effectivebarrier to current across the monolayer mediated by a redox ion speciesin an aqueous solution in contact with the monolayer, and binding of aligand-binding agent to the ligand on the monolayer surface is effectiveto measurably increase the current across of the monolayer mediated bysuch redox species, the apparatus further includes a chamber adapted tocontain such an aqueous solution of redox species in contact with saidmonolayer, and the detecting means includes circuit means for measuringion-mediated current across said monolayer, in response to bindingevents occurring between said receptor and ligand.
 6. The apparatus ofclaim 2, designed for gravimetric detection of binding of aligand-binding agent to the monolayer ligand, whereinthe biosensorsurface is a piezoelectric crystal, and the detecting means includesmeans for generating a surface acoustic wave in said crystal and meansfor detecting the shift in wave frequency, velocity, or resonancefrequency of the surface acoustic wave produced by binding ofligand-binding agent to said ligand.
 7. The apparatus of claim 2,designed for optical surface plasmon resonance (SPR) detection ofbinding of a ligand-binding agent to the monolayer ligand, whereinthebiosensor surface is a transparent dielectric substrate coated with athin metal layer on which said monolayer is formed, said substrate andmetal layer forming a plasmon resonance interface, and said detectingmeans includes means for exciting surface plasmons at a plasmonresonance angle that is dependent on the optical properties of the metalfilm and attached monolayer, and means for detecting the shift inplasmon resonance angle produced by binding of ligand-binding agent tosaid ligand.
 8. The apparatus of claim 2, designed for optical detectionof binding of a ligand-binding agent to the monolayer ligand,whereinsaid detecting means includes means for irradiating saidmonolayer with a light beam, and means for detecting a change in theoptical characteristics of the monolayer produced by binding ofligand-binding agent to said ligand.
 9. The apparatus of claim 1,wherein the first and second subunit peptides are oppositely charged.10. The apparatus of claim 1, wherein the detector surface includesfirst and second regions, each having a different selected ligandattached to the second peptide.